Abstract Two acetylcholinesterase (ace) genes have been reported in many insect species. In pests such as Helicoverpa assulta and Plutella xylostellas, ace1 gene encodes the predominant synaptic enzyme that is the main target of organophosphorus (OP) and carbamate pesticides. It has been reported that pesticide selection has an impact on the ace gene evolution. The domesticated silkworm, Bombyx mori, also has two ace genes. We studied ace gene expression and enzyme activities in silkworm as this has not faced pesticide selection over the past decades. The expression levels of two ace genes, Bm-ace1 and Bm-ace2, were estimated by quantitative real-time polymerase chain reaction. Bm-ace2 was expressed more highly than Bm-ace1 in all tested samples of different developmental stages or tissues, suggesting ace2, rather than ace1, is the major type of acetylcholinesterase (AChE) in Bombyx mori. This is inconsistent with the aforementioned lepidopterons agricultural pests, partly be due to the widespread use of pesticides that may induce high expression of the ace1 gene in these pests. Besides high expression in the head, Bm-ace1 also expresses highly in the silk glands and Bm-ace2 is abundant in the germline, implying both ace genes may have potential non-hydrolytic roles in development. Furthermore, we found that the mRNA levels of two ace genes and their ratios (ace2/ace1) change day to day in the first and third instars. This challenges the conventional method of estimating enzymatic activity using crude extract as an enzyme solution, as it is a mixture of AChE1 and AChE2. An efficient and simple method for separating different AChEs is necessary for reliable toxicological analyses.